S. aureus is an important human pathogen capable of causing serious, life-threatening infections and is one of the most common organisms to do so. This pathogen possesses multiple mechanisms by which it resists the killing effects of biocides and antibiotics, including overexpression of membrane-based proteins called multidrug resistance (MDR)-conferring efflux pumps (EPs). In fact, efflux is the single most important mechanism by which bacteria such as S. aureus can evade the effects of multiple structurally different antimicrobial agents simultaneously. EP activity also predisposes S. aureus to acquire target-based high level resistance-conferring mutations to some pump substrates by reducing intracellular concentrations to subinhibitory levels. EPs belong to one of five different protein families that are differentiated by structural characteristics and energy source used for substrate transport. The Multidrug and Toxic compound Extrusion (MATE) family is the most recently described and members are found not only in bacteria but also in eukaryotes including plants, yeast, and humans. Typical substrates for MATE pumps include mono- and bivalent organic cations such as biocides and disinfectants, fluoroquinolones, and anticancer agents. Acquisition of MDR S. aureus strains, including those with increased expression of MATE and other MDR efflux pump genes, can produce undesirable consequences such as prolonged hospital stays, increased healthcare costs, and most importantly increased morbidity and mortality. MepA is the first and only MATE MDR EP identified in S. aureus, and overexpression of mepA occurs in clinical strains. Point mutations in mepR, which encodes MepR, a MarR-family transcriptional repressor of mepA, that inactivate the protein frequently are the bases of mepA overexpression and are found in clinical strains and easily produced in the laboratory. However, other mechanisms of mepRA regulation also exist as mepA-overexpressing clinical strains lacking mepR mutations have been identified. This application proposes experiments designed to increase our understanding of the mepRA pump system in particular and MDR EPs of S. aureus in general. Our goals are to (1) Determine the details of MepR-DNA and MepR-inducer interactions by structural biology investigations and characterize the MepR-inducer binding site(s) using mutagenesis; (2) Determine the functional characteristics of the MepA pump and employ mutagenesis to better understand substrate/inhibitor interactions with it, which will inform the future structural biology analysis of the protein; (3) Characterize MepR-dependent and - independent mepRA regulatory mechanisms, including naturally-occurring MepR substitution and operator site mutations and trans-acting factors. MepR functional and operator site binding studies and analyses of plasmid libraries will be employed to accomplish this goal. The detailed study of MepA, combined with similar earlier work with other clinically important S. aureus MDR EPs (NorA and QacA/B), will help in the rational design of broad-spectrum EP inhibitors.